An example of an isozyme is glucokinase, a variant of hexokinase which is not
inhibited by glucose 6-phosphate. Its different regulatory features and lower
affinity for glucose (compared to other hexokinases), allows it to serve different
functions in cells of specific organs, such as

Both of these processes must only occur when glucose is abundant,or
problems occur.

Isozymes or Isoenzymes are proteins with different structure which catalyze
the same reaction. Frequently they are oligomers made with different
polypeptide chains, so they usually differ in regulatory mechanisms and in
kinetic characteristics.

From the physiological point of view, isozymes allow the existence of similar
enzymes with different characteristics, “customized” to specific tissue
requirements or metabolic conditions.

One example of the advantages of having isoenzymes for adjusting the
metabolism to different conditions and/ or in different organs is the following:

Glucokinase and Hexokinase are typical examples of isoenzymes. In fact,
there are four Hexokinases: I, II, III and IV. Hexokinase I is present in all
mammalian tissues, and Hexokinase IV, aka Glucokinase, is found mainly
in liver, pancreas and brain.

Both enzymes catalyze the phosphorylation of Glucose:

Glucose + ATP —–à Glucose 6 (P) + ADP

Hexokinase I has a low Km and is inhibited by glucose 6 (P). Glucokinase
is not inhibited by Glucose 6 (P) and his Km is high. These two facts
indicate that the activity of glucokinase depends on the availability
of substrate and not on the demand of the product.

Since Glucokinase is not inhibited by glucose 6 phosphate, in
conditions of high concentrations of glucose this enzyme
continues phosphorylating glucose, which can be used for
glycogen synthesis in liver. Additionally, since Glucokinase
has a high Km, its activity does not compromise the supply
of glucose to other organs; in other words, if Glucokinase
had a low Km, and since it is not inhibited by its product, it
would continue converting glucose to glucose 6 phosphate
in the liver, making glucose unavailable for other organs
(remember that after meals, glucose arrives first to the liver
through the portal system).

The enzyme Lactate Dehydrogenase is made of two (H-
and M-) sub units, combined in different Permutations
and Combinations depending on the tissue in which it
is present as shown in table,

Type

Composition

Location

LDH1

HHHH

Heart and Erythrocyte

LDH2

HHHM

Heart and Erythrocyte

LDH3

HHMM

Brain and Kidney

LDH4

HMMM

Skeletal Muscle and Liver

LDH5

MMMM

Skeletal Muscle and Liver

While isozymes may be almost identical in function
(defined by Michaelis constant, KM)

The sum of zwitterion charges result in identifyjng
difference inmigratiion toward the anode by gel
electrophoresis, and this forms the basis for the use
of isozymes as molecular markers.

To identify isozymes, a crude protein extract is made by
grinding animal or plant tissue with an extraction buffer,
and the components of extract are separated according
to their charge by gel electrophoresis.

They were classically purified by ion-exchange column
chromatography after first precipitation with ammonium
sulfate, followed by dialysis.

In cells, the immediate energy sources involve glucose oxidation. In anaerobic metabolism, the donor of the phosphate group is adenosine triphosphate (ATP), and the reaction is catalyzed via the hexokinase or glucokinase: Glucose +ATP-Mg²+ = Glucose-6-phosphate (ΔGo = – 3.4 kcal/mol with hexokinase as the co-enzyme for the reaction.).
In the following step, the conversion of G-6-phosphate into F-1-6-bisphosphate is mediated by the enzyme phosphofructokinase with the co-factor ATP-Mg²+. This reaction has a large negative free energy difference and is irreversible under normal cellular conditions. In the second step of glycolysis, phosphoenolpyruvic acid in the presence of Mg²+ and K+ is transformed into pyruvic acid. In cancer cells or in the absence of oxygen, the transformation of pyruvic acid into lactic acid alters the process of glycolysis.
The energetic sum of anaerobic glycolysis is ΔGo = -34.64 kcal/mol. However a glucose molecule contains 686kcal/mol and, the energy difference (654.51 kcal) allows the potential for un-controlled reactions during carcinogenesis. The transfer of electrons from NADPH in each place of the conserved unit of energy transmits conformational exchanges in the mitochondrial ATPase. The reaction ADP³+ P²¯ + H²–à ATP + H2O is reversible. The terminal oxygen from ADP binds the P2¯ by forming an intermediate pentacovalent complex, resulting in the formation of ATP and H2O. This reaction requires Mg²+ and an ATP-synthetase, which is known as the H+-ATPase or the Fo-F1-ATPase complex. Intracellular calcium induces mitochondrial swelling and aging. [12].
The known marker of monitoring of treatment in cancer diseases, lactate dehydrogenase (LDH) is an enzyme that is localized to the cytosol of human cells and catalyzes the reversible reduction of pyruvate to lactate via using hydrogenated nicotinamide deaminase (NADH) as co-enzyme.
The causes of high LDH and high Mg levels in the serum include neoplastic states that promote the high production of intracellular LDH and the increased use of Mg²+ during molecular synthesis in processes pf carcinogenesis (Pyruvate acid>> LDH/NADH >>Lactate acid + NAD), [13].
LDH is released from tissues in patients with physiological or pathological conditions and is present in the serum as a tetramer that is composed of the two monomers LDH-A and LDH-B, which can be combined into 5 isoenzymes: LDH-1 (B4), LDH-2 (B3-A1), LDH-3 (B2-A2), LDH-4 (B1-A3) and LDH-5 (A4). The LDH-A gene is located on chromosome 11, whereas the LDH-B gene is located on chromosome 12. The monomers differ based on their sensitivity to allosteric modulators. They facilitate adaptive metabolism in various tissues. The LDH-4 isoform predominates in the myocardium, is inhibited by pyruvate and is guided by the anaerobic conversion to lactate.
Total LDH, which is derived from hemolytic processes, is used as a marker for monitoring the response to chemotherapy in patients with advanced neoplasm with or without metastasis. LDH levels in patients with malignant disease are increased as the result of high levels of the isoenzyme LDH-3 in patients with hematological malignant diseases and of the high level of the isoenzymes LDH-4 and LDH-5, which are increased in patients with other malignant diseases of tissues such as the liver, muscle, lungs, and conjunctive tissues. High concentrations of serum LDH damage the cell membrane [11, 31].

Relation between LDH and Mg as Factors of Interest in the Monitoring and Prognoses of Cancer

Lactate Dehydrogenase (LDH) is ubiquitous in animals and
man, and it occurs in different organs of the body, each
region having a unique conformation of the subunits, but
the significance was once disputed. Perhaps the experiments
of Jakob and Monod on the lac 1 operon put to restany
notions that isoenzymes and their conformational forms are
something of no real significance. This concept does not
necessarily apply in all cases of isoenzyme differences, by
which I mean that there may be a difference in reactivity at
the active site.

For that matter, Jakob and Monod discovered and elucidated
allosterism.

The site the effector binds to is termed the allosteric site.
Allosteric sites allow effectors to bind to the protein, often
resulting in a conformational change. Effectors that enhance
the protein’s activity are referred to as allosteric activators,
whereas those that decrease the protein’s activity are calledallosteric inhibitors.

Allosteric regulations are a natural example of control loops,
such as feedback from downstream products or feedforward from upstream substrates. Long-range allostery is especially
important in cell signaling. Allosteric regulation
is also particularly important in the cell’s ability to adjustenzyme activity.

The term allostery comes from the Greekallos (ἄλλος), “other,”
and stereos (στερεὀς), “solid (object).” This is in reference
to the fact that the regulatory site of an allosteric protein is
physically distinct from its active site.

Jacob and Monod model of lac repressor

Most allosteric effects can be explained by the concertedMWC model put forth by Monod, Wyman, and Changeux, [2]
or by the sequential model described by Koshland, Nemethy,
and Filmer.[3] Both postulate that enzyme subunits exist in
one of two conformations, tensed (T) or relaxed (R), and
that relaxed subunits bind substrate more readily than
those in the tense state. The two models differ most in
their assumptions about subunit interaction and the pre-
existence of both states.

The sequential model (2) of allosteric regulation holds that subunits
are not connected in such a way that a conformational change in
one induces a similar change in the others. Thus, all enzyme
subunits do not necessitate the same conformation. Moreover,
the sequential model dictates that molecules of substrate
bind via aninduced fit protocol. In general, when a subunit
randomly collides with a molecule of substrate, the active site,
in essence, forms a glove around its substrate.

While such an induced fit converts a subunit from the tensed
state to relaxed state, it does not propagate the conformational
change to adjacent subunits. Instead, substrate-binding at
one subunit only slightly alters the structure of other
subunits so that their binding sites are more receptive to
substrate.
To summarize:

subunits need not exist in the same conformation

molecules of substrate bind via induced-fit protocol

conformational changes are not propagated to all
subunits

The discovery of morpheeins has revealed a previously
unforeseen mechanism to target universally essential
enzymes for species-specific drug design and discovery.
A morpheein-based inhibitor would function by binding
to and stabilizing the inactive morpheein form of the
enzyme, thereby shifting the equilibrium to favor that form (3).

Non-regulatory allostery

A non-regulatory allosteric site refers to any non-regulatory
component of an enzyme (or any protein), that is not itself
an amino acid. For instance, many enzymes require sodium
binding to ensure proper function. However, the sodium
does not necessarily act as a regulatory subunit; the sodium
is always present and there are no known biological processes
to add/remove sodium to regulate enzyme activity. Non-
regulatory allostery could comprise any other ions besides
sodium (calcium, magnesium, zinc), as well as other chemicals
and possibly vitamins.

Lactate and malate dehydrogenases

LDH is a key enzyme in glycolysis. Anaerobic glycolysis is theconversion of pyruvate into lactate acid in the absence
of oxygen. This pathway is important to glycolysis in two main
ways. The first is that

if pyruvate were to build up glycoysis

the generation of ATP would slow.

The second is anaerobic respiration

allows for the regeneration of NAD+ from NADH.

NAD+ is required when glyceraldehyde-3-phosphate
dehydrogenase oxidizes glyceraldehyde-3-phosphate in
glycolysis, which generates NADH. Lactate dehydrogenase
is responsible for the anaerobic conversion of NADH to
NAD+. Click here to see the residues which form
interactions with pyruvate in the Lactate Dehydrogenase
from Cryptosporidium parvum (2fm3). (Wikipedia)

Glycolysis ends with the synthesis of pyruvate. But, to be
self-functioning, it must end with lactate. Why? Anaerobic
means “without oxygen”. This is tantamount to saying
“without mitochondria”.

The mitochondria are especially adept at oxidizing
NADH to NAD+. NAD+ is needed to keep the glyceraldehyde-
3-PO4 dehydrogenase reaction functioning.

If glycolysis is to continue when no oxygen is present or in
short supply (as in a working muscle), an alternative means
of oxidizing NADH must occur.

Pyruvate has 2 metabolic fates:

it can either be converted into lactate or to acetyl-CoA .
Note that in animals and plants the electrons in NADH
are transferred to pyruvate which reduces the carbonyl
carbon in the pyruvate molecule to an alcohol. The
reaction is catalyzed by the enzyme lactate dehydrogenase.
Lactate (or L-lactate to be more precise) is thus a
“waste product”, since it has no metabolic fate other
than to be converted back into pyruvate in a reverse of
the forward reaction.

More importantly, the NAD+ feeds back to the glyceraldehyde-
3-PO4 dehydrogenase reaction, which allows glycolysis
to continue. Were it not for lactate formation, glycolysis
as a self-functioning pathway could not exist.

In yeast a slightly different end of glycolysis becomes apparent.
Yeast do not synthesize lactate. They do, however, oxidize
NADH back to NAD+ anaerobically. How do they do this? The
answer is they make ethanol. In the reaction the pyruvate is
converted into acetaldehyde. The reaction is catalyzed by a
lyase enzyme, pyruvate decarboxylase, which removes the
carboxyl group as a CO2. Acetaldehyde is formed because
the electron pair that bonds the –COO group is not removed
by the decarboxylation. A proton is plucked from the
environment giving the final product, acetaldehyde.
Acetaldehyde is now the substrate that will oxidize NADH to
NAD+ and in the process ethanol is formed.

There is another advantage to the pyruvate-lactate interchange.
The lactate formed by lactate dehydrogenase can be
reconverted. This allows a cell to synthesize glucose from lactate.
Converting lactate to glucose is a major feature of gluconeogenesis,
an anabolic pathway that synthesizes glucose from smaller
precursors such as lactate. This is important because acetyl-CoA
cannot be converted back to pyruvate and hence cannot be a
source of carbons for glucose biosynthesis.

ADP. ADP is required in the 3-phosphoglycerate kinase reaction
and in the pyruvate kinase reaction. It is formed from ATP in the
hexokinase reaction and the phosphofructokinase-I reaction.

NADH, ADP and PO4. NADH oxidation is important in glycolysis.
NADH is converted into NAD+ in the mitochondria. That
reaction is promoted by O2 ; NAD+ stays in the mitochondria.
Also in the mitochondria, ATP is formed by condensing ADP
with PO4. Thus, O2 allows mitochondria to out-compete the
cytosol for ADP, NADH and PO4, all limiting substrates or
coenzymes.

In vertebrates, gluconeogenesis takes place mainly in the liver
and, to a lesser extent, in the cortex of kidneys. In many
animals, the process occurs during periods of fasting,starvation, low-carbohydrate diets, or intense exercise.
The process is highly endergonic until it is coupled to the
hydrolysis of ATP or GTP, effectively making the processexergonic. For example, the pathway leading from pyruvate
to glucose-6-phosphate requires 4 molecules of ATP and
2 molecules of GTP to proceed spontaneously. Gluco-
neogenesis is a target of therapy for type II diabetes,
such as metformin, which inhibits glucose formation
and stimulates glucose uptake by cells.

Lactate is formed at the endstage of glycolysis with insufficient
oxygen is transported to the liver where it is converted intopyruvate by the Cori cycle using the enzyme lactate
dehydrogenase. In this reaction lactate loses two electrons
(becomes oxidized) and is converted to pyruvate. NAD+
gains two electrons (is reduced) and is converted to NADH.

Both lactate and NAD+ bind to the active site of the enzyme
lactate dehydrogenase and both lactate and NAD+ participate
in the catalysis reaction. In fact, catalysis could not occur
unless the coenzyme NAD+ bound to the active site.

As lactate concentration builds in the circulation from heavy
muscle production (M-type), or from circulatory insufficiency,
the circulating lactic acid reaches the liver.

The lactic acid is taken up by the liver, and the high
concentration of lactic acid drives the backward reaction,
unrestricted.

Pyruvate, the first designated substrate of the gluconeogenic
pathway, can then be used to generate glucose. Transamination
or deamination of amino acids facilitates entering of their
carbon skeleton into the cycle directly (as pyruvate or
oxaloacetate), or indirectly via the citric acid cycle. It is
known that odd-chain fatty acids can be oxidized to yieldpropionyl-CoA, a precursor for succinyl-CoA, which can
be converted to pyruvate and enter into gluconeogenesis.

Lactic dehydrogenase isozymes of bovine and rabbit lens and
cornea were analyzed by starch gel electrophoresis.
Although there was a progressive loss of enzyme activity in
the lenses of both species with increasing age, the loss of
isozymes was more clearly evident in the bovine lens. In
the adult bovine lens,

lactic dehydrogenase isozyme Iwas predominant,

while in the adult rabbit lens, isozymes 3–5were mainly present.

The mobility of lens isozymes was identical to that of isozymes
in other tissues. Furthermore, the isozymes were not localized
to any major specific lens crystallin.

Changes in the activity of lactate dehydrogenase (LDH) (l-lactate:
NAD+ oxidoreductase EC 1.1.1.27) isozymes are associated with
the growth and differentiation of bovine lens cells. Calf and adult
lens epithelial cells contain all 5 isozymes. The cathodal forms are
most active in the calf-epithelial cells; the anodal forms are most
active in the fiber cells. This transition from cathodal to anodal
forms of lactate dehydrogenase in the epithelial cells is associated
with cellular aging.

During the differentiation of an epithelial cell to a fiber cell, in calf
and adult lenses there is an enhancement of

the transition from cathodal forms to anodal forms.

The regulation of lactate dehydrogenase subunit synthesis may
be associated, therefore, with

the replicative activity of these cells.

In cells having the greatest replicative activity (calf epithelial
cells) the cathodal isozymes are most active; in cells having a
decreased mitotic activity (adult epithelial cells) the anodal
isozymes are most active. The non-replicative

fiber cell of calf and adult shows a transition toward the
anodal forms.

Although lens fiber cells have a low rate of oxidative metabolismlactate dehydrogenase-I is the most active isozyme in these
cells. Kinetically,

lactate dehydrogenase-I factors other than, or in addition
to, the regulation of carbohydrate metabolism

are involved in regulating the synthesis of lactate dehydrogenase subunits.

Abbreviations LDH; lactate dehydrogenase

What is not examined to resolve the discrepancy (see the next item):

The Vessell paper was a challenge to the work in Nathan
Kaplan’s lab. However, there is sufficient complexity revealed
in these works that there is no conceptual foundation.

The analogy is to the loss of cell nuclei in crystallin lens
fiber formation with the LDH-H type subunits (aerobic?)

The findings are reproduced in several laboratories.

In the lens, glucose is catabolized primarily to lactic
acid, and is not appreciably combusted to CO2
(J Kinoshita. Glucose metabolism of Lens)

However, synthetic processes, including nuclear DNA and
cell replication requires TPNH. This is produced by means
of the Pentose Shunt.

The most favorable conditions for the lens are achieved
by incubating in a medium containing glucose in the
presence of oxygen. Under these conditions of
incubation (Kinoshita)

the lens remains completely transparent,

it maintains normal levels of high energy phosphate
bonds and cations, and

it shows a high rate of arginine incorporationinto protein.

incubation in the absence of glucose, but in the presence of oxygen

a haze is found in the lens,

a drop in high energy phosphate level is observed, and

Changes in cation levels are apparent.

A 50 percent decrease in the incorporation of arginine
into lens protein is also observed.

the most unfavorable condition for the lens is an anaerobic
incubation in a medium without glucose

Pirie2 observed that a-glycerophosphate is one of the end products
of lens metabolism. Its oxidation with DPN as the cofactor could
channel its electrons directly into the ETC to produce energy without
involving the Krebs cycle. a-Glycerophosphate is formed from intermediates of the glycolytic scheme by reduction of dihydroxy-
acetone phosphate, one of the triose phosphates produced in
glycolysis.

the dehydrogenase of the mitochondria catalyzes the transfer
of elections to form DPNH by the following reactions:

a-glycerophosphate + DPN+ ± dihydroxyacetone ……..

phosphate + DPNH.

The DPNH is channeled into the oxidative phosphorylation
mechanism to form ATP. The dihydroxyacetone phosphate
then diffuses out into the soluble cytoplasm, interacts with
the glycolytic intermediates by the reversal of the above reaction,

The activity of lactate dehydrogenase (LDH) and its isoenzyme
pattern were studied in four concentric layers of adult
bovine and calf lenses. In both groups the specific activity of
the total LDH diminished progressively toward the internal
nuclear layer; the decrease was greater in the adult lenses.
The enzyme activities in the cortical layers of the calf lens
were lower than in the adult lens, but in the inner nuclear layers,
the opposite was found. All of the 5 LDH isoenzymes were found
in each layer. In both groups of animals the LDH1 isoenzyme
prevailed, followed by LDH2. No differences were found in the
percentage of each isoenzyme in the different lens layers.
The differences in the activitie(s) of LDH found may be due

to post-translational or post-synthetic modifications which
may occur during the aging process.

Lactate dehydrogenase (LDH) interconverts pyruvate and
lactate with concomitant interconversion of NADH and NAD(+).
Although crystal structures of a variety of LDH have previously
been described, a notable absence has been any of the
three known human forms of this glycolytic enzyme. We have
now determined the crystal structures of two isoforms of
human LDH-the M form, predominantly found in muscle; and
the H form, found mainly in cardiac muscle. Both structures
have been crystallized as ternary complexes in the presence
of the NADH cofactor and oxamate, a substrate-like inhibitor.

Although each of these isoforms has different kinetic properties,
the domain structure, subunit association, and active-site regions
are indistinguishable between the two structures.

The pK(a) that governs the K(M) for pyruvate for the two isozymes
is found to differ by about 0.94 pH units, consistent with variation in
pK(a) of the active-site histidine.

The close similarity of these crystal structures suggests the distinctive
activity of these enzyme isoforms is likely to result

directly from variation of charged surface residues peripheral to the active site,

a hypothesis supported by electrostatic calculations based on each structure.

The catalytic step in the conversion of pyruvate to L-lactate in the
enzyme L-lactate dehydrogenase involves the transfer of both a
proton and a hydride ion (A.R. Clarke, T. Atkinson and J.J. Holbrook,
TIBS, 14 (1989) 101.) However, it is not known whether the
reaction is concerted or, if a multistep process, the order in
which the transfers of the proton and the hydride ions take
place. Four possible non-concerted mechanisms can be
proposed, which differ in the order of the transfers of the
proton and hydride ion and the protonation state of the substrate
carboxylate group during the transfers. The energies and
optimized geometries of the corresponding intermediates,
protonated pyruvate, protonated pyruvic acid, deprotonated
L-lactate and deprotonated L-lactic acid, are computed using
the semiempirical AM 1 and ab initio SCF/3–21 G – methods.
These calculations are complementary to the study of
the substrates for the enzyme discussed in a previous paper
(K.E. Norris and J.E. Gready, J. Mol. Struct. (Theochem),
258 (1992) 109.) The structures and energetics of protonated
pyruvate and deprotonated L-lactate provide some
important insights into the requirements for enzymic reaction
and the characteristics of the transition state.

Pyruvate production by Enterococcus casseliflavus A-12
from gluconate in an alkaline medium

A newly isolated lactic acid bacterium, Enterococcus casseliflavus
A-12, produced pyruvic acid (16 g/l) during aerobic culture in
an alkaline medium containing sodium gluconate (50 g/l) as
the carbon source. The production was dependent on the pH
of the culture, the optimum initial pH being 10.0. With static
culture, the organism produced lactic acid (2.7 g/l) from both
gluconate and glucose. Pyruvate did not accumulate in growing
cultures on glucose, but resting cells obtained from a culture
on gluconate produced pyruvate from glucose as well as
gluconate. The enzyme profiles of the organism, which
grew on gluconate and glucose, suggested that gluconate
was metabolized via the Entner-Doudoroff and Embdem-
Meyerhof-Parnas pathways in aerobic culture, and that glucose
was oxidized mainly via the latter pathway under both aerobic
and anaerobic conditions. Gluconokinase, a key enzyme in
the aerobic metabolism of gluconate, was partially purified
from this strain and characterized.

A specific, highly active malate dehydrogenase by redesign
of a lactate dehydrogenase framework

Three variations to the structure of the nicotinamide adenine
dinucleotide (NAD)-dependent L-lactate dehydrogenase
from Bacillus stearothermophilus were made to try to
change the substrate specificity from lactate to malate:
Asp197—-Asn, Thr246—-Gly, and Gln102—-Arg).

Each modification shifts the specificity from lactate to malate, although

only the last (Gln102—-Arg) provides an effective and
highly specific catalyst for the new substrate.

This synthetic enzyme has a ratio of catalytic rate (kcat) to
Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1,

equal to that of native lactate dehydrogenase for its natural
substrate, pyruvate, and a maximum velocity (250 s-1),
which is double that reported for a natural malate from B.
stearothermophilus.

Malate dehydrogenase (MDH) (EC 1.1.1.37) catalyzes the
conversion of oxaloacetate and malate. This reaction is
important in cellular metabolism, and it is coupled with
easily detectable cofactor oxidation/reduction. It is a
rather ubiquitous enzyme, for which several isoforms
have been identified, differing in their subcellular
localization and their specificity for the cofactor NAD
or NADP. The nucleotide binding characteristics can
be altered by a single amino acid change. Multiple
amino acid sequence alignments of MDH show there is a

low degree of primary structural similarity, apart from
several positions crucial for catalysis, cofactor binding
and the subunit interface.

Despite the low amino acids sequence identity their
3-dimensional structures are very similar.

MDH is a group of multimeric enzymes consisting of
identical subunits usually organized as either dimer
or tetramers with subunit molecular weights of 30-35 kDa.

Malate dehydrogenase (MDH2) is an enzyme in the citric
acid cycle that catalyzes the conversion of malate into
oxaloacetate (using NAD+) and vice versa (this is a
reversible reaction). Malate dehydrogenase is also
involved in gluconeogenesis, the synthesis of glucose
from smaller molecules.Pyruvate in the mitochondria is acted upon by pyruvate
carboxylase to form oxaloacetate, a citric acid cycle
intermediate.In order to get the oxaloacetate out of the mitochondria,
malate dehydrogenase reduces it to malate, and it then
traverses the inner mitochondrial membrane.Once in the cytosol, the malate is oxidized back to
oxaloacetate by cytosolic malate dehydrogenase.

Malate Dehydrogenase (MDH)(PDB entry 2x0i) is most known
for its role in the metabolic pathway of the tricarboxylic acid cycle,
critical to cellular respiration; The enzyme has other metabolic roles in –

glyoxylate bypass,

amino acid synthesis,

glucogenesis, and

oxidation/reduction balance .

An oxidoreductase, MDH has been extensively studied due to its
isozymes The enzyme exists in two places inside a cell:

the mitochondria and cytoplasm.

In the mitochondria, the enzyme catalyzes the reaction of
malate to oxaloacetate;

in the cytoplasm, the enzyme catalyzes oxaloacetate to
malate to allow transport.

The enzyme malate dehydrogenase is composed of either
a dimer or tetramer depending on the location of the enzyme
and the organism it is located in. During catalysis, the enzyme
subunits are

non-cooperative between active sites.

The mitochondrial MDH is complexly,

allosterically controlled by citrate, but no other known
metabolic regulation mechanisms have been discovered.

the exact mechanism of regulation has yet to be discovered.

Kinetically, the pH of optimization is 7.6 for oxaloacetate
conversion and 9.6 for malate conversion. The reported
K(m) value for malate conversion is 215 uM and the V(max)
value is 87.8 uM/min.

Comment:

The mMDH and the cMDH both form ternary complex
of MDH-NAD+-OAA formed during the forward reaction,
like the LDH H-type isozyme LDH-NAD+-PYR (mot the M-type).
However, the binding of the Enz-coenzyme-substrate is not
as strong as for the H-type LDH. .The regulatory role has
not been established.

ABSTRACT These studies determine the levels of malate
dehydrogenase isoenzymes in cardiac muscle by a steady
state kinetic method which depends on the differential inhibition
of these isoenzyme forms by high concentrations of oxaloacetate.
This inhibition is similar to that exhibited by lactate dehydrogenase
in the presence of high concentrations of pyruvate. The results
obtained by this method are comparable in resolution to those
obtained by CM-Sephadex fractionation and by differential
centrifugation for the analyses of mitochondrial malate
dehydrogenase and cytoplasmic malate dehydrogenase in
tissues. The use of standard curves of percent inhibition of
malate dehydrogenase activity plotted against the ratio of
mitochondrial MDH activity to the total of mMDH and cMDH
activities [ malate dehydrogenase ratio] (percent m-type) is
introduced for studies of comparative mitochondrial
function in heart muscle of different species or in different
tissues of the same species.

Using a reconstituted [3H]norepinephrine
release assay in permeabilized PC12 cells, we
found that essential proteins that support the triggering
stage of Ca21-stimulated exocytosis are enriched in an
EGTA extract of brain membranes. Fractionation of this
extract allowed purification of two factors that stimulate
secretion in the absence of any other cytosolic proteins.
These are calmodulin and protein kinase Ca
(PKCa). Their effects on secretion were confirmed using
commercial and recombinant proteins. Calmodulin enhances
secretion in the absence of ATP, whereas PKC
requires ATP to increase secretion, suggesting that
phosphorylation is involved in PKC- but not calmodulin
mediated stimulation. Both proteins modulate release
events that occur in the triggering stage of exocytosis.

Department of Biochemistry, University College of Science,
Osmania University, Hyderabad 500 007, India

The maintenance of regular vascular tone substantially
depends on the bioavailability of endothelium-derived
nitric oxide (NO) synthesized by eNOS. The essential
role of NO, as the elusive endothelium-derived relaxing
factor (EDRF), was the topic of research that won the
1998 Nobel Prize in Physiology or Medicine. The eNOS
gene, as a candidate gene in the investigations on
hypertension genetics, has attracted the attention of
several researchers because of the established role
of NO in vascular homeostasis. The eNOS variants
located in the 7q35-q36 region have been investigated
for their association with CVD, particularly hypertension.
Three variants, viz., (i) G894T substitution in exon 7
resulting in a Glu to Asp substitution at codon 298 (rs1799983),
(ii) an insertion-deletion in intron 4 (4a/b) consisting of two
alleles (the a*-deletion which has four tandem 27-bp repeats
and the b*-insertion having five repeats), and (iii) a T786C
substitution in the promoter region (rs2070744), have been
extensively studied20-22. Individual SNPs often cause only
a modest change in the resulting gene expression or function.
It is, therefore, the concurrent presence of a number of SNPs
or haplotypes within a defined region of the chromosome that
determines susceptibility to disease development and progression,
particularly in case of polygenic diseases.

Shankarishan et al24 analysed for the first time the prevalence
of eNOS exon 7 Glu298Asp polymorphism in tea garden community
of North Eastern India, who are a high risk group for CVD. This study
also included indigenous Assamese population and found no
significant difference between the distribution patterns of eNOS
exon 7 Glu298Asp variants between the communities. They have
rightly mentioned that for developing public health policies and
programmes it is necessary to know the prevalence and distribution
of the candidate genes in the population, as well as trends in
different population groups. They have also observed that the
eNOS exon 7 homozygous GG wild genotype (75.8%) was
predominant in the study population followed by heterozygous
GT genotype (21.5%) and homozygous TT genotype (2.7%).
The frequency distribution of the homozygous GG, heterozygous
GT and homozygous mutant TT genotypes were comparable to
that of the north Indian and south Indian population.

Polymorphisms in the endothelial nitric oxide synthase gene have
been associated inconsistently with cardiovascular diseases.
Varying distribution of eNOS variants among ethnic groups may
explain inter-ethnic differences in nitric oxide mediated vasodilation
and response to drugs28. Different population studies showed
association of eNOS polymorphisms with variations in NO
formation and response to drugs. Cardiovascular drugs including
statins increase eNOS expression and upregulate NO formation
and this effect may be responsible for protective, pleiotropic
effects produced by statins31. With respect to hypertension,
studies have reported interactions between diuretics and
polymorphisms in eNOS gene. Particularly, the Glu298Asp
polymorphism made a statistically significant contribution to
predicting blood pressure response to diuretics.

Neuronal Nitric Oxide Synthase and Its Interaction
With Soluble Guanylate Cyclase Is a Key Factor for
the Vascular Dysfunction of Experimental Sepsis

Vascular dysfunction plays a central role in sepsis, and it is
characterized by hypotension and hyporesponsiveness to
vasoconstrictors. Nitric oxide is regarded as a central element
of sepsis vascular dysfunction. The high amounts of nitric
oxide produced during sepsis are mainly derived from the
inducible isoform of nitric oxide synthase 2.
We have previously shown that nitric oxide synthase 2 levels
decrease in later stages of sepsis, whereas levels and activity
of soluble guanylate cyclase increase. Therefore, we studied
the putative role of other relevant nitric oxide sources, namely,

the neuronal (nitric oxide synthase 1) isoform, in sepsis

and its relationship with soluble guanylate cyclase.

We also studied the consequences of

nitric oxide synthase 1 blockade in the hyporesponsiveness
to vasoconstrictors.

1) Both nitric oxide synthase 1 and soluble guanylate cyclase
are expressed in higher levels in vascular tissues during sepsis;

3) pharmacological blockade of nitric oxide synthase 1 using
7-nitroindazole or S-methyl-l-thiocitrulline reverts the hypo
responsiveness to phenylephrine and increases the vaso
constrictor effect of norepinephrine and phenylephrine.

Sepsis induces increased expression and physical association
of nitric oxide synthase 1/soluble guanylate cyclase and a higher
production of cyclic guanosine monophosphate that together
may help explain sepsis-induced vascular dysfunction.

†Department of Chemistry, Department of Molecular Biosciences,
Chemistry of Life Processes Institute, Center for Molecular Innovation
and Drug Discovery, Northwestern University, Evanston, Illinois
‡Departments of Molecular Biology and Biochemistry, Pharmaceutical
Sciences, and Chemistry, University of California, Irvine, California,
Department of Biochemistry, University of Texas Health Science Center,
San Antonio, Texas

Overproduction of NO by nNOS is implicated in the pathogenesis of
diverse neuronal disorders. Since NO signaling is involved in
diverse physiological functions, selective inhibition of nNOS
over other isoforms is essential to minimize side effects. A series of
α-amino functionalized aminopyridine derivatives (3−8) were
designed to probe the structure−activity relationship between ligand,
heme propionate, and H4B. Compound 8R was identified as the
most potent and selective molecule of this study, exhibiting a Ki of
24 nM for nNOS, with 273-fold and 2822-fold selectivity against
iNOS and eNOS, respectively.Although crystal structures of 8R
complexed with nNOS and eNOS revealed a similar binding mode,
the selectivity stems from the distinct electrostatic environments in
two isoforms that result in much lower inhibitor binding free energy
in nNOS than in eNOS. These findings provide a basis for further
development of simple, but even more selective and potent, nNOS
inhibitors

In cells, the immediate energy sources involve glucose oxidation. In anaerobic metabolism, the donor of the phosphate group is adenosine triphosphate (ATP), and the reaction is catalyzed via the hexokinase or glucokinase: Glucose +ATP-Mg²+ = Glucose-6-phosphate (ΔGo = – 3.4 kcal/mol with hexokinase as the co-enzyme for the reaction.).
In the following step, the conversion of G-6-phosphate into F-1-6-bisphosphate is mediated by the enzyme phosphofructokinase with the co-factor ATP-Mg²+. This reaction has a large negative free energy difference and is irreversible under normal cellular conditions. In the second step of glycolysis, phosphoenolpyruvic acid in the presence of Mg²+ and K+ is transformed into pyruvic acid. In cancer cells or in the absence of oxygen, the transformation of pyruvic acid into lactic acid alters the process of glycolysis.
The energetic sum of anaerobic glycolysis is ΔGo = -34.64 kcal/mol. However a glucose molecule contains 686kcal/mol and, the energy difference (654.51 kcal) allows the potential for un-controlled reactions during carcinogenesis. The transfer of electrons from NADPH in each place of the conserved unit of energy transmits conformational exchanges in the mitochondrial ATPase. The reaction ADP³+ P²¯ + H²–à ATP + H2O is reversible. The terminal oxygen from ADP binds the P2¯ by forming an intermediate pentacovalent complex, resulting in the formation of ATP and H2O. This reaction requires Mg²+ and an ATP-synthetase, which is known as the H+-ATPase or the Fo-F1-ATPase complex. Intracellular calcium induces mitochondrial swelling and aging. [12].
The known marker of monitoring of treatment in cancer diseases, lactate dehydrogenase (LDH) is an enzyme that is localized to the cytosol of human cells and catalyzes the reversible reduction of pyruvate to lactate via using hydrogenated nicotinamide deaminase (NADH) as co-enzyme.
The causes of high LDH and high Mg levels in the serum include neoplastic states that promote the high production of intracellular LDH and the increased use of Mg²+ during molecular synthesis in processes pf carcinogenesis (Pyruvate acid>> LDH/NADH >>Lactate acid + NAD), [13].
LDH is released from tissues in patients with physiological or pathological conditions and is present in the serum as a tetramer that is composed of the two monomers LDH-A and LDH-B, which can be combined into 5 isoenzymes: LDH-1 (B4), LDH-2 (B3-A1), LDH-3 (B2-A2), LDH-4 (B1-A3) and LDH-5 (A4). The LDH-A gene is located on chromosome 11, whereas the LDH-B gene is located on chromosome 12. The monomers differ based on their sensitivity to allosteric modulators. They facilitate adaptive metabolism in various tissues. The LDH-4 isoform predominates in the myocardium, is inhibited by pyruvate and is guided by the anaerobic conversion to lactate.
Total LDH, which is derived from hemolytic processes, is used as a marker for monitoring the response to chemotherapy in patients with advanced neoplasm with or without metastasis. LDH levels in patients with malignant disease are increased as the result of high levels of the isoenzyme LDH-3 in patients with hematological malignant diseases and of the high level of the isoenzymes LDH-4 and LDH-5, which are increased in patients with other malignant diseases of tissues such as the liver, muscle, lungs, and conjunctive tissues. High concentrations of serum LDH damage the cell membrane [11, 31].

Relation between LDH and Mg as Factors of Interest in the Monitoring and Prognoses of Cancer

The inhibition be pyruvate is related by a ternary complex formed by NAD+ formed in the catalytic forward reaction Pyruvate + NADH –> Lactate + NAD(+). The reaction can be followed in an Aminco-Morrow stop-flow analyzer and occurs in ~ 500 msec. The reaction does not occur with the muscle type LDH, and it is regulatory in function. I did not know about the role of intracellular Mg(2+) in the catalysis, as my own work was in Nate Kaplan’s lab in 1970-73.

This difference in the behavior of the isoenzyme types was considered to be important then in elucidating functional roles, but it was challenged by Vessell earlier. The isoenzymes were first described by Clement Markert at Yale. I think, but don’t know, that the Mg++ would have a role in driving the forward reaction, but I can’t conceptualize how it might have any role in the difference between muscle and heart.

I didn’t quite know why oncologists used it specifically. Cancer cells exhibit the reliance on the anaerobic (muscle) type enzyme, which is also typical of liver, but with respect to the adenylate kinases – the liver AK and muscle AK (myokinase) are different. That difference was discovered by Masahiro Chiga, and differences in the reaction with sulfhydryl reagents were identified by Percy Russell.

Oddly enough, Vessell had a point. The RBC has the heart type predominance, not the M-type. He thought that it was related to the loss of nuclei from the reticulocyte. I did not buy that, and I had worked on the lens of the eye at the time.

The IDH1 and IDH2 genes are mutated in > 75% of different malignant diseases. Two distinct alterations are caused by tumor-derived mutations in IDH1 or IDH2,
IDH1 and IDH2 mutations have been observed in myeloid malignancies, including de novo and secondary AML (15%–30%), and in pre-leukemic clone malignancies, including myelodysplastic syndrome and myeloproliferative neoplasm (85% of the chronic phase and 20% of transformed cases in acute leukemia.
Aurelian Udristioiu, M.D
City Targu Jiu, Romania
AACC, NACB, Member, USA.